Anti-cardiolipin IgG autoantibodies associate with circulating extracellular DNA in severe COVID-19

Whereas the detection of antiphospholipid autoantibodies (aPL) in COVID-19 is of increasing interest, their role is still unclear. We analyzed a large aPL panel in 157 patients with COVID-19 according to the disease severity. We also investigated a potential association between aPL and extracellular DNA (exDNA, n = 85) or circulating markers of neutrophil extracellular traps (NET) such as citrullinated histones H3 (CitH3, n = 49). A total of 157 sera of patients infected by SARS-CoV-2 were collected. A large aPL panel including lupus anticoagulant, anti-cardiolipin and anti-beta-2 glycoprotein I (IgG, IgM and IgA), anti-phosphatidylethanolamine IgA, anti-prothrombin (IgG and IgM) was retrospectively analyzed according to the disease severity. We found a total aPL prevalence of 54.8% with almost half of the cases having aCL IgG. Within an extended panel of aPL, only aCL IgG were associated with COVID-19 severity. Additionally, severe patients displayed higher CitH3 levels than mild patients. Interestingly, we highlighted a significant association between the levels of aCL IgG and exDNA only in aCL positive patients with severe disease. In conclusion, we showed a significant link between aPL, namely aCL IgG, and circulating exDNA in patients with severe form of COVID-19, that could exacerbate the thrombo-inflammatory state related to disease severity.


Scientific Reports
| (2022) 12:12523 | https://doi.org/10.1038/s41598-022-15969-y www.nature.com/scientificreports/ risk factors of severe illness during COVID-19 infection 13 . Importantly, they can contribute to the generation of a thrombo-inflammatory state, as observed in acute respiratory distress syndrome, or in cancer 14,15 . As 5-15% of patients suffering from COVID-19, particularly the elderly and people with preexisting cardiovascular diseases, develop severe pneumonia and coagulopathy disorders 16,17 , we hypothesized that an autoimmune mechanism would exacerbate the inflammatory response and contribute to excessive exDNA or NET production. In line with this, we recently described the presence of antiphospholipid autoantibodies (aPL) in COVID-19 patients, by showing that anti-cardiolipin IgG autoantibodies (aCL) are highly and independently associated with COVID-19 severity 18 . aPL represent a large family of autoantibodies that are central for the diagnosis of antiphospholipid syndrome (APS), an autoimmune-associated coagulopathy, and are also considered as vascular risk factors 19 . In APS, thrombotic effects have been mainly associated with aPL-activated cells such as endothelial cells or platelets. aPL can stimulate neutrophils to produce exDNA and NETs in APS patients 20 . Similarly, purified IgG fraction isolated from COVID-19 patients with aPL has been shown to promote NETosis when incubated with neutrophils purified from healthy subjects 21 . Several studies reported the presence of aPL in COVID-19 patients, but conclusions should be clarified about their frequency, type and clinical effects. To this end, we retrospectively assessed in 157 patients with COVID-19 a large aPL panel. It includes lupus anticoagulant (LA), aCL IgG/IgM/IgA, anti-beta-2 glycoprotein I (aB2GPI) IgG/IgM/IgA, anti-phosphatidylethanolamine (aPE) IgG/IgM, and anti-prothrombin (aPT) IgG/IgM autoantibodies. To further investigate the pathological role of aPL produced in COVID-19, we measured circulating exDNA and CitH3 levels, and analyzed them according to aPL detection and disease severity.

Material and methods
Patients. All patients with COVID-19 were confirmed to be infected by SARS-CoV-2 by real-time reverse transcriptase PCR (RT-qPCR) testing or by a positive serology for anti-SARS CoV-2 IgG.
aPL dosage was performed on serum samples collected from 157 COVID-19 patients admitted between March 2020 and July 2021 in the University Hospital of Marseilles (France) and consecutively referred to the hospital laboratory for immunological exploration.
Clinical data were collected from the day of sampling. They included co-morbidities (history of thrombosis, history of stroke, coronary heart disease, hypertension, diabetes, heart failure and chronic respiratory disease), clinical presentation (mild or severe), and duration of symptoms.
Definitions. "Severe clinical presentation" was defined based on at least one of the following criteria: respiratory rate > 30 cycles/min, oxygen saturation ≤ 93%, PaO 2 /FiO 2 ratio ≤ 300 mmHg, shock (defined by the need of vasopressors) or respiratory failure requiring the admission to intensive care units (ICUs) 22 . "Thrombosis" was defined as the formation of a thrombus within a blood vessel (artery or vein) confirmed by ultrasound, magnetic resonance imaging (MRI) or X-ray Computed Tomography (CT). "Chronic respiratory disease" included chronic obstructive pulmonary disease, asthma, or lung cancer. "Heart failure" included class III or class IV stages according NYHA classification. "Acute respiratory distress syndrome" was defined according to Berlin definition 23 . Treatments. The patients received anticoagulant treatment with prophylactic heparin consisting of subcutaneous low molecular weight heparin (enoxaparin 4000 IU/day) or subcutaneous unfractionated heparin (5000 IU every 12 h) if patients had a clearance < 30 mL/min or if admitted to ICU. Combination of non-invasive ventilation/high flow oxygen or mechanical ventilation were used when respiratory support was needed.

ICU control group.
To assess the specificity of aPL in COVID-19, patients admitted to ICU with a negative SARS-CoV-2 RT-qPCR and/or absence of anti-SARS CoV-2 IgG, and screened for aPL in ICU during the study period, were analyzed according to their etiology of ICU hospitalization.
Ethics. All serum samples collected from patients infected by SARS-CoV-2 were part of a declared Biobank (DC 2020-4028) in compliance with ethical directives. For ICU non-COVID-19 patients, samples were part of a declared Biobank (DC 2012-1704) in compliance with ethical directives. Informed consent was obtained from all participants. This study received approval from the national review board Comité de Protection des Personnes Ile de France XI (20027-60604, March 25th 2020) and fulfilled local requirements in terms of data collection and protection of data (RGPD/APHM 2020-80). This study was conducted according to the Declaration of Helsinki.
Biological tests. aCL and aB2GPI levels were measured by ELISA with Cardiolisa Theradiag (Marne la Vallée, France) and Orgentec Diagnostica (Mainz, Germany), respectively. Positive cut-off were set-up respectively at 15 U/mL and 8 U/mL according to manufacturers' recommendations and on-site validation. To avoid non-specific binding issues, each positive sample was duplicated and serum non-specific background of uncoated well was subtracted from the measured optical density (OD) of coated well. To assess cofactor dependence of aCL IgG, all positive sera were tested with another ELISA assay (AIDA, Bad Kreuznach, Germany) with manufacturers cut-off equals to 15 U/mL. aPT were detected with an in-house ELISA previously described 24 . ΔOD for each sample was calculated by subtracting OD of coated well from OD obtained with non-coated well. The aPT levels were reported as a ratio of ΔOD of patient/ΔOD of a selected control serum with a ΔOD at the cut-off value. The result is positive when the ratio is above 1.  25 . The optical density (OD) of each well was measured at 405 nm and the OD of non-coated well was subtracted for each sample (ΔOD). These cut-off values were 0.47 and 0.68 ΔOD, corresponding to the following arbitrary units: 18 and 59 U/mL for aPE IgG and aPE IgM, respectively.
Quantification of exDNA levels in serum was performed according to manufacturer's instructions using the Quant-iT PicoGreen DNA assay kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) as previously published 26 .
Quantification of CitH3 levels in serum was performed according to manufacturer's instructions using Citrullinated Histone H3 (Clone 11D3) ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA).

Statistical analysis.
Analysis was performed using R version 3.03 (R Development Core Team) and Graph-Pad Prism V6.05 (GraphPad Software, La Jolla, CA, USA). Data are described as Mean ± standard deviation in the tables. Shapiro-Wilk test was used to test for data normality and two-tailed Student t test was used to test variable differences between groups. Pearson's Chi-squared test was used to test difference in frequencies between groups for categorical variables. Correlations between markers were evaluated using Pearson correlation analysis. Significance level was set at 0.05. The study was conducted in accordance to the STROBE statement.

Characteristics of patients with COVID-19. A total of 157 patients infected by SARS-CoV-2 were
included in this study ( Fig. 1, Table 1), with 53 hospitalized in ICU. The mean age of patients was 68 ± 16 years and 57% of them were males. According to the clinical presentation at sampling time, this cohort was divided into two groups: mild (n = 59) and severe (n = 98), as defined above.
No differences in terms of age, gender, and co-morbidities were observed between the two groups. In contrast, the duration of symptoms was longer in the severe group than in the mild group (p < 0.001). The patients in the severe group were more often anticoagulated (p = 0.001) and invasively ventilated (p < 0.001) than those in the mild group. Fatal evolution was significantly associated with severity (p = 0.007).
Antinuclear autoantibodies. ANA detection by IIF was performed in 105 patients from our cohort.

Anti-cardiolipin IgG characterization and clinical association.
Taking into account the methodological issues on ELISA assays and the lack of agreement between methods, we wanted to confirm the aCL positivity of 29 patients. We used an ELISA using beta-2 glycoprotein I as a sole cofactor source (assay 2) and found that 62% of the 29 positive aCL patients were also positive by using assay 2.
To further analyze aCL IgG association with the severe form of COVID-19, we also analyzed aCL IgG with clinical characteristics and biomarkers (Table 4). In addition to the disease severity, a significant association of aCL IgG positivity was found with symptom duration (p < 0.001), transfer to ICU (p < 0.001) and invasive ventilation (p < 0.001). No association was found with in-hospital mortality, clinical history of patients or thrombotic events occurring during the active phase of the disease. Concerning the biomarkers, we observed one significant association between aCL IgG and elevated polymorphonuclear neutrophils (PMN) counts (p = 0.007).
Anti-cardiolipin IgG and circulating extracellular DNA association. Since exDNA have been recently proposed as markers of COVID-19 complications, we investigated a potential association between aCL and exDNA. From the 85 patients tested, we found a significant association between aCL IgG positivity and exDNA levels in serum (Table 4). Indeed, circulating exDNA levels were significantly higher in aCL IgG positive patients than in aCL IgG negative ones (p = 0.031, Table 4). There was weak correlation between exDNA and aCL IgG levels (Pearson's r = 0.216, n = 85, p = 0.047) and no association between disease severity and circulating exDNA levels considering the whole population of patients (Fig. 3a). However, among patients with severe COVID-19, exDNA levels were significantly higher (p = 0.014) in aCL IgG positive patients than in IgG aCL negative ones (Fig. 3b). Table 3. Characteristics of patients admitted in ICU with aPL positivity. aPL antiphospholipid, aCL anticardiolipin, aB2GPI anti-beta-2 glycoprotein I autoantibodies, ICU intensive care unit, ARDS acute respiratory distress syndrome, n number.  www.nature.com/scientificreports/ samples). We found a significant association (p = 0.024) between disease severity and NETs levels (Fig. 3c). Regarding the severe group of patients for which 32 samples were available, no difference was observed in NETs levels according to aCL positivity, in contrast to exDNA levels (Fig. 3d).

Discussion
Our study showed an association between aPL and circulating exDNA in patients with severe form of COVID-19, that could be involved in the exacerbation of the thrombo-inflammatory response associated with the disease severity. Several studies reported the presence of aPL in COVID-19, but contradictory results have been found regarding the prevalence, type and clinical relevance of aPL. In our study, we found a total aPL prevalence of 55% with almost half of positive aCL IgG and showed that, within an extended panel of aPL, aCL IgG were associated with severe forms of the disease. This result confirmed our previous data performed on 56 patients with COVID-19 for which only conventional aPL had been sought 18 .
The search for LA was difficult to interpret because the patients affected by COVID-19 received anticoagulant therapy. Devreese et al. found 23% of patients positive for aCL and/or aB2GPI and more than 50% of patients positive for LA 27 , whereas Cristiano et al. described a lower percentage of positivity of around 5% and 2% for aCL and/or aB2GPI and LA, respectively 28 . In accordance with our data, Zuo et al. found a total prevalence of more than 50% in their cohort of 172 patients with COVID-19, with a predominance of aCL 21 . Trahtemberg et al. also corroborated our data by showing that aCL are predominant and associated with disease severity 29 . This heterogeneity of results could be interrelated to the methodology used and/or population studied. Due to the difficult interpretation of aPL positivity, we systematically controlled positive samples in duplicate, and, to address the specificity, the absorbance of uncoated wells treated in the same conditions was systematically subtracted to avoid noise from nonspecific binding. Moreover, in this study, aCL IgG positivity was confirmed by using another ELISA assay and showed that majority of aCL IgG were beta-2-glycoprotein I cofactor dependent, as described for pathogenic aPL in APS 30 . In addition, to test whether aPL could be generated by ARDS or www.nature.com/scientificreports/ septic shock, a control cohort of 122 ICU patients, showing that aPL positivity, mostly for aCL, was significantly associated with COVID-19 and not with non-COVID-19 related-ARDS or related-septic shock. In our study, the presence of aCL was rather associated with inflammation than thrombosis. Indeed, most of patients were on anticoagulant medication, and we did not have controls before the infection. It is therefore difficult to discuss about the association between aCL and thrombosis. Nevertheless, this finding invites to propose a follow-up of patient's aCL positivity, especially since the persistence of various symptoms in patients who recovered from COVID-19 was recently defined as long COVID or post-covid syndrome (PCS) 31 . In PCS, persistent neurological symptoms have been described, and we can note that neurological disorders are also described in APS 32 .
In contrast to autoantibodies directed against cardiolipin, no association between ANA and disease severity was established. We showed the presence of ANA in COVID-19, without identifying any particular antibody specificity. ANA are useful biomarkers for the diagnosis and the monitoring of autoimmune rheumatic diseases. Since it has been reported that ANA could precede by several years a symptomatic autoimmune disease 33 , a follow-up of patients could also be advocated.
Interestingly we showed that NETs detected by CitH3 but not exDNA, are associated with severe form of COVID-19. In contrast, only exDNA levels were found significantly higher in severe COVID-19 patients with aCL IgG positivity than in aCL IgG negative ones. This significant association was not observed for CitH3, possibly because of the low number of patients tested for CitH3. In this line, a major limitation of our study was missing data in CitH3 and exDNA because of insufficient quantity of serum for some patients. However patient characteristics of the exDNA or CitH3 cohorts were comparable to those of the total cohort (Supplementary Table S1), which validate the results. Additionally, the possible difference in origin of exDNA and CitH3 could also explain the difference in results. Nevertheless, our data led us to propose a potential role of aPL in COVID-19: aCL would exacerbate the severity of the disease by affecting exDNA release. ExDNA and other DAMPs molecules, such as CitH3, are major structural elements of NETs. ExDNA release also reflects tissue damage and cell apoptosis. Therefore cells expressing ACE2, the entry receptor for SARS-CoV-2, are potential sources of exDNA during COVID-19 infection. ExDNA represents relevant markers of inflammation 14,15 and have been proposed in the pathogenesis of inflammatory and autoimmune diseases, such as APS 20 . More recently it has been shown that their levels increased during the evolution www.nature.com/scientificreports/ of the COVID-19 and thus, exDNA are proposed as biomarkers for the patient outcome 11 . Altogether, our data reinforced the link between exDNA release and auto-immunity in COVID-19. Pathogenicity of aPL from patients with COVID-19 has also been previously revealed by the fact that injection of IgG fractions isolated from these patients accelerated thrombotic events in an animal model of venous thrombosis, and importantly promoted NET release by human neutrophils 21 . One can speculate that aPL could activate apoptosis of other cell types such as endothelial cells or other cells expressing ACE2.We can thus hypothesize that autoimmunity enhances the deleterious effect of the inflammatory response, through aCL and exDNA release. Since oxidative stress is known to play a critical role in cell death 34 , increase of production of reactive oxygen species (ROS) has been associated with ExDNA release, and we can speculate that ROS formation could be associated with severe damage in COVID-19. In agreement, Wenzhong et al. showed by employing bioinformatics methods that SARS-CoV-2 generates ROS by iron capture and therefore damages the human immune system 35 . The preferential involvement of autoantibodies against cardiolipin in the severity of the disease may be related to a new described mechanism showing that aCL recognize a cell surface complex composed of lysobiphosphatidic acid (LBPA) and endothelial protein C receptor (EPCR) 36 . Subsequent endocytosis activates Toll like receptor 7 and 8 (TLR 7 and TLR8) and type I IFN signaling, leading to further synthesis of autoantibodies and ROS production. As TLR7 and type I IFN signaling are involved in COVID-19, we can hypothesize that aCL generated during the infection could signal through the recently described EPCR-LBPA pathway involved in inflammation and thrombosis. A putative mechanism of aCL role in severe forms of COVID-19 is proposed in Fig. 4.
In conclusion, we propose that severe forms of COVID-19 engage an autoimmune mechanism that may exacerbate inflammatory pathways through exDNA release. Our results have two major applications in the management of COVID-19: the follow-up of patients by the detection of aCL and a proposal of an original therapeutic strategy targeting extracellular DNA.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.